Synthetic peptides useful in biological essays for detecting infections caused by group O HIV-1 viruses

ABSTRACT

Synthetic peptides of the monomer type with 13 to 33 amino acids, in linear form or in a form cyclized by means of inter-cysteine disulphide bridges, have the general formula (I): 
 
Δ-Z-TrpGlyCys-Θ-CysTyrThrSer-Ω  (I) 
 
wherein Δis a biotinyl radical, a biocytinyl radical, a hydrogen atom, an acetyl (CH 3 CO—) radical, an aliphatic chain which may contain one or two thiol, an aldehyde functional group, or an amine functional group, Z represents peptide sequence -   1 -Ser-   2 -, -   1 -Gln-   2 -, or -   1 -Asn-   2 -, wherein -   1  represents a peptide sequence of 0 to 9 amino acids and -   2  represents a peptide sequence of 0 to 5 amino acids, Θis -Arg Gly Arg Leu Ile-(SEQ ID NO: 15), -Arg Gly Arg Leu Val-(SEQ ID NO: 16), -Arg Gly Lys Leu Ile-(SEQ ID NO: 17), -Arg Gly Lys Leu Val-(SEQ ID NO: 18), -Lys Gly Arg Leu Ile-(SEQ ID NO: 19), or -Lys Gly Arg Leu Val-(SEQ ID NO: 20), Ω, attached to the —CO— group of serine, is a hydroxyl (—OH) radical, an amino (—NH 2 ) radical, an alkoxy radical having 1 to 6 carbon atoms, a peptide sequence of formula Val-Σ-Ψ wherein Σ represents a sequence of formula -(AA 1 )-Trp Asn-(AA 2 )-(AA 3 ) wherein (AA 1 ) represents an amino acid different from lysine, (AA 2 ) represents an amino acid, and (AA 3 ) is serine or a threonine residue, and Ψ, attached to the —CO— residue of the free AA 3  amino acid, is OH, NH 2 , or an alkoxy radical having from 1 to 6 carbon atoms, and a peptide sequence of formula -Val-Ψ wherein Ψ, attached to the —CO— residue of valine, is OH, NH 2 , or an alkoxy radical having from 1 to 6 carbon atoms.

The invention relates to synthetic peptides which can be used inbiological tests for the detection of infections due to the group OHIV-1 viruses, to the method for preparing them, to compositions andkits containing such peptides and to the biological tests using suchpeptides.

Group O HIV-1 retroviruses are known in the prior art. Patent EP0,345,375 and patent application EP 0,657,532 describe the ANT 70 andANT 70 NA isolates isolated from Cameroonian patients. These documentsdescribe more precisely antigens and antigenic compositions containinglysates or proteins of these isolates, the nucleic acids correspondingto the genomic RNA, hybridization methods using these nucleic acids,methods of producing the isolates indicated above as well as methods ofpreparing p12, p16, p25, gp41 and gp120 proteins of these retroviruses.

Application EP 0,591,914 describes the MVP 5180/91 isolate. Thisisolate, characterized by Western blotting, exhibits, like the previousisolate, differences in relation to the HIV-1 retrovirus isolates whichhave been known for a long time. Application EP 0,591,914 describesprecisely the DNA sequence of the MVP 5180/91 isolate and indicatesprecisely the location of the gag, pol and env genes. Application EP0,591,914 further describes synthetic peptides of the V3 loop as well asthe immunodominant region (gp41). They are useful for biological tests,in particular for the in vitro detection of group 0 HIV-1 antibodies.

Application EP 0,673,948 describes synthetic or recombinant peptidesconsisting of 15 to 50 amino acids (M) and comprising the sequence-VWGIRQLRARLQALETLIQ NQQRLNLWGXKGKLIXYTSVKWNTSWSGR- in which Xrepresents either a cysteine residue, or a serine residue. Thesepeptides are useful in the diagnostic field for the detection ofinfections due to certain group O HIV-1 retrovirus isolates.

Application EP 0,727,483 is also known which describes the MVP 2901/94isolate which also forms part of the retroviruses belonging to the groupO HIV-1 family. This application describes certain antigens havingwell-determined peptide sequences. These peptide sequences correspond topart of the sequence of gp120 and part of gp41 (immunodominant region)of the MVP 2901/94 isolate.

Application WO 96/12809 describes two new isolates belonging to thegroup O HIV-1 family. They are the VAU and DUR isolates. Thisapplication describes certain peptide sequences derived from the twoviruses cited above, which are useful for the detection of antibodiesrecognizing the HIV-1 VAU or DUR peptide sequences.

Application WO 96/32293 describes two antigens derived from the sequenceof the ANT 70 isolate. They are the antigen called MDL061 and theantigen MDL056, of the immunodominant region of gp41. According to thisinvention, to detect 100% of the samples of a limited collection of serafrom patients infected with the group O HIV-1 virus, it is necessary touse compositions containing these two peptides, since each isolatedpeptide does not allow, on its own, satisfactory results to be obtained.

Indeed, it is practically impossible, in the light of the geneticvariability shown by the isolates of the group O virus, to guaranteeserological screening of individuals infected by the use of antigensderived from the same and sole isolate. This means that it is notpossible to obtain reagents which guarantee 100% sensitivity. The Ogroup thus raises, for the first time, a major problem; it is theinability of certain serological reagents to recognize individualsinfected with particularly divergent groups or subtypes. This isprecisely the case for the group O HIV-1 viruses.

Application WO 96/40763 also stresses the great divergence of the ogroup. This application describes peptides which incorporate, into anatural HIV-1 type B sequence, a few minor modifications (replacement ofone or two amino acids). According to this application, these hybridpeptides are capable of reacting with anti-group O antibodies.

Application WO 96/27013 describes a series of new group O HIV-1 virusesdesignated BCF 01, BCF 02, BCF 03, BCF06, BCF 07, BCF 08, BCF09, BCF11,BCF12, BCF13 and BCF14 as well as a series of peptides of thecorresponding gp41 dominant region which are called ESS/BCF02,FAN/BCF01, LOB/BCF06, MAN/BCF07, NKO/BCF08, POC/BCF03, NAN/BCF11, BCF09,BCF12, BCF13 and BCF14. A number of these peptides are difficult tohandle in diagnosis because of their low solubility, especially thepeptide BCF13.

Unexpectedly, it has now been found that certain synthetic peptides arediagnostic reagents of superior quality and they make it possible tosatisfactorily screen patients infected with group O HIV-1 retroviruses.These peptides are composed of variable sequences articulated aroundhighly conserved short sequences, which are present in isolates of thegroup O HIV-1 retroviruses. The peptides of the invention make itpossible to obtain results which are quite superior to those obtainedwith synthetic peptides carrying immunodominant epitopes of the gp41(env) of certain group O HIV-1 isolates.

Subsequently, to name the amino acids, the three-letter nomenclaturewill be used.

The synthetic peptides of the invention correspond to the generalformula (I):Δ-Z-TrpGlyCys-Θ-CysTyrThrSer-Ω  (I)in which:

Δ represents a biotinyl radical, a biocytinyl radical, a hydrogen atom,an acetyl (CH₃CO—) radical, an aliphatic chain which may contain one ortwo thiol, aldehyde or amine functional groups, the aliphatic chainpreferably being an alkyl chain of 1 to 6 carbon atoms or an alkenylchain of 2 to 6 carbon atoms, or an aminoalkylcarbonyl chain of 2 to 6carbon atoms,

Z represents a peptide sequence of one of the formulae (II) to (X):-

₁-Ser-

₂-  (II)-Ser-

₂-  (III)-

₁-Ser-  (IV)-

₁-Gln-

₂-  (V)-Gln-

₂-  (VI)-

₁-Gln-  (VII)-

₁-Asn-

₂-  (VIII)-Asn-

₂-  (IX)

₁-Asn-  (X)

in which:

-   -   ₁ represents a peptide sequence of 0 to 9 amino acids and    -   ₂ represents a peptide sequence of 0 to 5 amino acids,

Θ represents a peptide sequence of formula (XI):-(AA₁)-(AA₂)-(AA₃)-(AA₄)-(AA₅)-  (XI)

-   -   in which:    -   (AA₁) represents either a lysine residue, or an arginine        residue, or an ornithine residue,    -   (AA₂) represents either a glycine residue, or an asparagine        residue,    -   (AA₃) represents either a lysine residue, or an arginine        residue, or an ornithine residue,    -   (AA₄) represents either a leucine residue, or an alanine        residue, or an isoleucine residue, or a glutamine residue,    -   (AA₅) represents either an isoleucine residue, or a valine        residue, or a leucine residue, or a threonine residue, or a        norleucine residue, or a norvaline residue,    -   provided, however, that (AA₁), (AA₂), (AA₃), (AA₄) and (AA₅)        never form together the peptide sequences -Lys Gly Lys Leu lie-        and -Lys Gly Lys Leu Val-,

Ω, attached to the —CO— group of serine, represents:

-   -   a hydroxyl (—OH) radical or an amino (—NH₂) radical,    -   an alkoxy radical comprising from 1 to 6 carbon atoms,    -   a peptide sequence of formula (XII):        -Val-E-YT (XII)    -   in which Σ represents a sequence of formula (XIII) or of formula        (XIV):        -(AA₆)-Trp Asn-(AA₇)-(AA₈)  (XIII)        -(AA₆)-Trp His-(AA₇)-(AA₈)  (XIV)    -   in which:        -   (AA₆) represents an amino acid different from lysine,        -   (AA₇) represents an amino acid,        -   (AA₈) represents a serine or threonine residue, and Ψ,            attached to the —CO— residue of the free AA₈ amino acid,            represents an OH or NH₂ group or an alkoxy radical            comprising from 1 to 6 carbon atoms,    -   a peptide sequence of formula (XV):        -Val-Ψ  (XV)    -   in which Ψ, attached to the —CO— residue of valine, has the same        meaning as for the formula (XII),    -   or a peptide sequence of one of the formulae (XVI) to (XVIII)        -Z-TrpGlyCys-Θ-CysTyrThrSer-T  (XVI)        Val-Σ-Z-TrpGlyCys-Θ-CysTyrThrSerVal-Σ-Ψ  (XVII)        Val-Z-TrpGlyCys-Θ-CysTyrThrSerVal-Ψ  (XVIII)    -   in which Z and Θ have the definition given for the formula (I)        and Σ has the definition given for the-formula (XII) and Ψ,        attached to the —CO— residue of serine, on the —CO— residue of        the AA₈ amino acid or on the —CO— residue of valine, has the        same meaning as for the formula (XII).

When Ω represents a peptide sequence of one of the formulae (XVI) to(XVIII), the peptide of formula (I) becomes a dimer whose size may varyfrom 26 to 66 amino acids. When Ω does not represent a peptide sequenceof one of the formulae (XVI) to (XVIII), the peptides of formula (I) areof the monomeric type and their size may vary from 13 to 33 amino acids.

The peptides according to the invention may be either in a linear form,or in a form cyclized by means of inter-cysteine disulphide bridges.

The compounds of formula (I) in which (AA₅) represents either a valineresidue, or a leucine residue, or a threonine residue are preferred, andwhen Q corresponds to a peptide sequence of formula (XII), (AA₆)represents either a glutamine residue or an arginine residue.

The peptides of formula (I) are preferred in which:

Δ represents a biotinyl radical, a hydrogen atom or an aliphatic chainwhich may contain one or two thiol, aldehyde or amine functional groups,the aliphatic chain preferably being an alkyl chain of 1 to 6 carbonatoms, or an aminoalkylcarbonyl chain of 2 to 6 carbon atoms,

Z represents a peptide sequence of formula (II) or (V), in which

₁ represents a peptide sequence of two amino acids and

₂ represents an amino acid, or a sequence of formula (IV), in which

₁ represents three amino acids, or a peptide sequence of formula (VIII,in which

₁ represents a peptide sequence of nine, eight or three amino acids and

₂ a peptide sequence of five amino acids,

Θ represents a peptide sequence of formula:-Lys Gly Arg Leu Val-,-Arg Gly Lys Ala Val-,Arg Gly Arg Leu Val-,or-Arg Gly Arg Ala Val-,andΩ represents a hydroxyl group, the peptide sequence (XV) or one of thefollowing sequences which correspond to the peptide sequence of formula(XII):-Val Arg Trp Asn Glu Thr-Ψ,-Val Gin Trp Asn Glu Thr-Ψor-Val Gin Trp Asn Ser Thr-Ψ.

Preferably, Z represents a peptide sequence of formula:

-   -Leu Leu Ser Ser--   -Leu Leu Asn Ser--   -Leu Leu Gin Ser--   -Arg Leu Asn Ser--   -Ala Leu Glu Thr Leu Leu Gin Asn Gln Gln Leu Leu Asn Ser--   -Ala Leu Glu Thr Leu Leu Gln Asn Gln Gln Leu Leu Asp Leu--   -Ala Leu Glu Thr Leu Leu Gin Asn Gln Gln Leu Leu Asn Ile--   -Leu Asn Gln Gln Arg Leu Leu Asn Ser--   or-   -Arg Ala Leu Glu Thr Leu Leu Asn Gln Gln Arg Leu Leu Asn Ser-

Also forming part of the invention are the synthetic peptides comprisingfrom 20 to 50 amino acids and corresponding to the formula (Ia):Δ-Z_(a)-TrpGlyCys-Θ-CysTyrThrSer-Ω_(a)  (Ia)in which Z_(a) represents a radical of formulae IIa to Xa:

_(1a)-Ser-

_(2a)  (IIa)-Ser-

_(2a)  (IIIa)-

_(1a)-Ser  (IVa)

_(1a)-Gln-

_(2a)  (Va)-Gln-

_(2a)  (VIa)

_(1a)-Gln-  (VIIa)

_(1a)-Asn-

_(2a)  (VIIIa)-Asn-

_(2a)  (IXa)-

_(1a)-Asn  (Xa)in which:

-   -   _(1a) represents a peptide sequence of 1 to 5 amino acids and        _(2a) an amino acid,    -   Ω_(a) represents a peptide sequence of formula (XII), as defined        for the formula (I), or a peptide sequence of formula (XVIIa):        Val-Σ-Z_(a)-TrpGlyCys-Θ-CysTyrThrSerVal-Σ-Ψ  (XVIIa)    -   in which Z_(a) has the meaning given for the formula (Ia) and    -   Δ, Θ, Σ and Ψ have the same meaning as for the formula (I).

The peptides of formula (I) or (Ia) including one of the followingsequences (these peptides may be of the dimer type or of the monomertype as defined above) are preferred. The sequences are given accordingto the one- and three-letter nomenclatures: Sequence No. 1-LLSLWGCRGKAVCYTSVQWNET- or -Leu Leu Ser Leu Trp Gly Cys Arg Gly Lys AlaVal Cys Tyr Thr Ser Val Gln Trp Asn  1              5                   10                   15                  20Glu Thr-     22 Sequence No. 2 -LLSLWGCRGRLVCYTSVQWNET- or -Leu Leu SerLeu Trp Gly Cys Arg Gly Arg Leu Val Cys Tyr Thr Ser Val Gln Trp Asn  1              5                   10                   15                  20Glu Thr-     22 Sequence No. 3 -LLSSWGCKGRLVCYTSVQWNET- or -Leu Leu SerSer Trp Gly Cys Lys Gly Arg Leu Val Cys Tyr Thr Ser Val Gln Trp Asn  1              5                   10                   15                  20Glu Thr-     22 Sequence No. 4 -LLSSWGCKGRLVCYTSVQWNST- or -Leu Leu SerSer Trp Gly Cys Lys Gly Arg Leu Val Cys Tyr Thr Ser Val Gln Trp Asn  1              5                   10                   15                  20Ser Thr-     22 Sequence No. 5 -LLQSWGCKGRLVCYTSVQWNST- or -Leu Leu GlnSer Trp Gly Cys Lys Gly Arg Leu Val Cys Tyr Thr Ser Val Gln Trp Asn  1              5                   10                   15                  20Ser Thr-     22 Sequence No. 6 -LLNSWGCRGKAVCYTSVQWNET- or -Leu Leu AsnSer Trp Gly Cys Arg Gly Lys Ala Val Cys Tyr Thr Ser Val Gln Trp Asn  1              5                   10                   15                  20Glu Thr-     22 Sequence No. 7 -LLSLWGCRGRAVCYTSVQWNET- or -Leu Leu SerLeu Trp Gly Cys Arg Gly Arg Ala Val Cys Tyr Thr Ser Val Gln Trp Asn  1              5                   10                   15                  20Glu Thr-     22 Sequence No. 8 -LLSSWGCRGRLVCYTSVQWNET- or -Leu Leu SerSer Trp Gly Cys Arg Gly Arg Leu Val Cys Tyr Thr Ser Val Gln Trp Asn  1              5                   10                   15                  20Glu Thr-     22 Sequence No. 9: -LLSSWGCKGRLVCYTS- or -Leu Leu Ser SerTrp Gly Cys Lys Gly Arg Leu Val Cys Tyr Thr Ser-  1                5                 10                  15 Sequence No.10: -LLNSWGCKGRLVCYTS- or -Leu Leu Asn Ser Trp Gly Cys Lys Gly Arg LeuVal Cys Tyr Thr Ser-  1                5                 10                  15 Sequence No.11 -ALETLLQNQQLLNSWGCRGRLVCYTSVRWNET- or -Ala Leu Glu Thr Leu Leu GlnAsn Gln Gln Leu Leu Asn Ser Trp Gly Cys Arg Gly  1               5                  10                  15 Arg Leu ValCys Tyr Thr Ser Val Arg Trp Asn Glu Thr-20                  25                  30 Sequence No. 12:-ALETLLQNQQLLNIWGCRGRLVCYTSVRWNET- or -Ala Leu Glu Thr Leu Leu Gln AsnGln Gln Leu Leu Asn Ile Trp Gly Cys Arg Gly  1               5                  10                  15 Arg Leu ValCys Tyr Thr Ser Val Arg Trp Asn Glu Thr-20                  25                  30 Sequence No. 13:-ALETLLQNQQLLDLWGCRGRLVCYTSVRWNET- or -Ala Leu Glu Thr Leu Leu Gln AsnGln Gln Leu Leu Asp Leu Trp Gly Cys Arg Gly  1               5                  10                  15 Arg Leu ValCys Tyr Thr Ser Val Arg Trp Asn Glu Thr-20                  25                  30 Sequence No. 14:-LNQQRLLNSWGCKGRLVCYTSV- or -Leu Asn Gln Gln Arg Leu Leu Asn Ser Trp GlyCys Lys Gly Arg Leu Val Cys Tyr  1               5                  10                  15 Thr Ser Val-20 Sequence No. 15: -RALETLLNQQRLLNSWGCKGRLVCYTSV- or -Arg Ala Leu GluThr Leu Leu Asn Gln Gln Arg Leu Leu Asn Ser Trp Gly Cys Lys  1               5                  10                  15 Gly Arg LeuVal Cys Tyr Thr Ser Val- 20                  25 Sequence No. 16:-RLNSWGCKGRLVCYTSV- or -Arg Leu Asn Ser Trp Gly Cys Lys Gly Arg Leu ValCys Tyr Thr Ser Val-  1               5                  10                  15

The synthetic peptides below are particularly preferred peptides:PEPTIDE No. 1 (2B): SEQ ID No. 1 LLSLWGCRGKAVCYTSVQWNET or Leu Leu SerLeu Trp Gly Cys Arg Gly Lys Ala Val Cys Tyr Thr Ser Val Gln Trp Asn 1               5                   10                  15                  20Glu Thr     22 PEPTIDE No. 2 (3B): SEQ ID No. 2 LLSLWGCRGRLVCYTSVQWNETor Leu Leu Ser Leu Trp Gly Cys Arg Gly Arg Leu Val Cys Tyr Thr Ser ValGln Trp Asn 1               5                   10                  15                  20Glu Thr     22 PEPTIDE No. 3 (4B): SEQ ID No. 3 LLSSWGCKGRLVCYTSVQWNETor Leu Leu Ser Ser Trp Gly Cys Lys Gly Arg Leu Val Cys Tyr Thr Ser ValGln Trp Asn 1               5                   10                  15                  20Glu Thr     22 PEPTIDE No. 4 (5B): SEQ ID No. 4 LLSSWGCKGRLVCYTSVQWNSTor Leu Leu Ser Ser Trp Gly Cys Lys Gly Arg Leu Val Cys Tyr Thr Ser ValGln Trp Asn 1               5                   10                  15                  20Ser Thr     22 PEPTIDE No. 5 (6B): SEQ ID No. 5 LLQSWGCKGRLVCYTSVQWNSTor Leu Leu Gln Ser Trp Gly Cys Lys Gly Arg Leu Val Cys Tyr Thr Ser ValGln Trp Asn 1               5                   10                  15                  20Ser Thr     22 PEPTIDE No. 6: SEQ ID No. 6 LLNSWGCRGKAVCYTSVQWNET or LeuLeu Asn Ser Trp Gly Cys Arg Gly Lys Ala Val Cys Tyr Thr Ser Val Gln TrpAsn 1               5                   10                  15                  20Glu Thr     22 PEPTIDE No. 7: SEQ ID No. 7 LLSLWGCRGRAVCYTSVQWNET or LeuLeu Ser Leu Trp Gly Cys Arg Gly Arg Ala Val Cys Tyr Thr Ser Val Gln TrpAsn 1               5                   10                  15                  20Glu Thr     22 PEPTIDE No. 8 (7B): SEQ ID No. 8 LLSSWGCRGRLVCYTSVQWNETor Leu Leu Ser Ser Trp Gly Cys Arg Gly Arg Leu Val Cys Tyr Thr Ser ValGln Trp Asn 1               5                   10                  15                  20Glu Thr     22 PEPTIDE No. 9 (12B): SEQ ID No. 9 LLSSWGCKGRLVCYTS or LeuLeu Ser Ser Trp Gly Cys Lys Gly Arg Leu Val Cys Tyr Thr Ser 1              5                  10                 15 PEPTIDE No. 10(14B): SEQ ID No. 10 LLNSWGCKGRLVCYTS or Leu Leu Asn Ser Trp Gly Cys LysGly Arg Leu Val Cys Tyr Thr Ser 1              5                  10                 15 PEPTIDE No. 11(18B): SEQ ID No. 11 ALETLLQNQQLLNSWGCRGRLVCYTSVRWNET or Ala Leu Glu ThrLeu Leu Gln Asn Gln Gln Leu Leu Asn Ser Trp Gly Cys Arg Gly 1              5                  10                 15 Arg Leu Val CysTyr Thr Ser Val Arg Trp Asn Glu Thr20                  25                  30 PEPTIDE No. 12 (19B): SEQ IDNo. 12 ALETLLQNQQLLNIWGCRGRLVCYTSVRWNET or Ala Leu Glu Thr Leu Leu GlnAsn Gln Gln Leu Leu Asn Ile Trp Gly Cys Arg Gly 1              5                  10                 15 Arg Leu Val CysTyr Thr Ser Val Arg Trp Asn Glu Thr20                  25                  30 PEPTIDE No. 13 (20B): SEQ IDNo. 13 ALETLLQNQQLLDLWGCRGRLVCYTSVRWNET or -Ala Leu Glu Thr Leu Leu GlnAsn Gln Gln Leu Leu Asp Leu Trp Gly Cys Arg Gly 1              5                  10                 15 Arg Leu Val CysTyr Thr Ser Val Arg Trp Asn Glu Thr20                  25                  30 PEPTIDE No. 14 (21B): SEQ IDNo. 14 LNQQRLLNSWGCKGRLVCYTSV or Leu Asn Gln Gln Arg Leu Leu Asn Ser TrpGly Cys Lys Gly Arg Leu Val Cys Tyr 1              5                  10                 15 Thr Ser Val 20PEPTIDE No. 15 (22B): SEQ ID No. 15 RALETLLNQQRLLNSWGCKGRLVCYTSV or ArgAla Leu Glu Thr Leu Leu Asn Gln Gln Arg Leu Leu Asn Ser Trp Gly Cys Lys 1              5                  10                 15 Gly Arg Leu ValCys Tyr Thr Ser Val 20                  25 PEPTIDE No. 16 (23B): SEQ IDNo. 16 RLNSWGCKGRLVCYTSV or Arg Leu Asn Ser Trp Gly Cys Lys Gly Arg LeuVal Cys Tyr Thr Ser Val 1              5                  10                 15

The synthetic peptides of formula (I), which are the subject of thepresent invention, can be obtained by solid phase synthesis according toconventional methods: R. B. Merrifield, J. Amer. Chem. Soc. (1963), 83,pp. 2149-2154; R. C. Sheppard, in “Peptides 1971”, Nesvadba H. (ed.)North Holland, Amsterdam, pp. 111; E. Atherton and R. L. Sheppard, in“Solid phase peptide synthesis, a practical approach”, IRL PRESS,(1989), Oxford University Press, pp. 25-34. As automatic synthesizer, itis possible to use the synthesizer “9050 Plus Pep Synthesizer” fromMillipore or an equivalent synthesizer.

The solid support used for the syntheses should be compatible with thetechnique and the chemistry used. For example, for a synthesis on thesynthesizer “9050 Plus pep. Synthesizer”, it is recommended to use aresin suitable for the so-called “continuous flow” technique; the PEG PSresins meet these criteria. These supports consist of an arm (“spacer”)based on polyethylene glycol (PEG) situated between the functional groupof the polystyrene beads and the point of attachment of the first aminoacid. The nature of this point of anchorage may vary according to theC-terminal functional group chosen. In the present case, various PEG-PSresins were used.

The starting resin and the amino acids used as raw materials areproducts which are commercially available (PerSeptive-Biosystem, orNeosystem).

For the peptide synthesis, the following side chain protecting groupswere used: Amino acids Protecting group ArgininePentamethyl-2,2,4,6,7-dihydrobenzofuran-5- sulphonyl (Pbf) Asparagine,Glutamine Trityl (Trt) Cysteine Trityl (Trt) or Acetamidomethyl (Acm)Serine, Threonine, tert-Butyl ether (tBu) Tyrosine Lysine, Tryptophantert-Butyloxycarbonyl (Boc)

The temporary protection of the primary amine functional group at the αposition of the amino acids which is used is the 9-fluorenylmethyloxylcarbonyl (Fmoc) group. The deprotection is carried out with a 20%solution of piperidine in dimethylformamide.

For the coupling, an excess of diisopropylcarbodiimide (DIPCDI) and1-hydroxybenzotriazole (HOBt) is preferably used.

After synthesis, the resin is washed with organic solvents(dimethylformamide, followed by dichloromethane), dried under vacuum andthen treated with a trifluoroacetic acid (TFA)-based solution cooled to0° C. and containing appropriate “scavengers”. There may be used, forexample, the K reagent containing 82% of trifluoroacetic acid, 5% ofphenol, 5% of water, 5% of thioanisole and 3% of ethanedithiol.

After filtration of the resin, the synthetic peptides are precipitatedand rinsed with ether.

The synthetic peptides are then purified by reversed phase liquidchromatography and their purity is determined by mass spectrometry. Assolid phase, it is possible to use, for example, the Bondapak C-18phase. The peptides are eluted by forming a linear gradient between twobuffer solutions, the first which is essentially aqueous (for examplewater-TFA 0.1%) and the second which is rather organic (for example amixture containing 60% acetonitrile, 39.92% water and 0.08% TFA). Thepure fractions collected are combined, concentrated under vacuum andfreeze-dried.

For the cyclization, the purified synthetic peptides are dissolved in anammonium acetate solution (10 mM). The pH is adjusted to 8.5 by additionof 1 M ammonium hydroxide. The solution is vigorously stirred. Thecyclization is complete after 18 hours. The pH is then reduced to 6 byaddition of acetic acid. The cyclized peptides are freeze-dried and thenpurified by reversed phase liquid chromatography as described above.

The immunoreactivity of the peptides of the invention was evaluated withthe aid of sera from patients predominantly of Cameroonian origininfected with group O HIV-1 retroviruses. The various tests carried outdemonstrated that the peptides of the invention, alone or in combination(compositions of peptides), make it possible to detect 100% of the serainfected with group O HIV-1 retroviruses.

The synthetic peptides of the invention therefore find application inimmunological tests for the screening of infections due to group O HIV-1retroviruses. It is also possible to use combinations of severalsynthetic peptides of formula 1. These combinations, which may containtwo or more peptides of formula 1, also form part of the invention.Combinations containing peptides No. 1 (2B) and No. 3 (4B) arepreferred.

It is also possible to use synthetic peptides of formula (I) of thepresent invention in combination with group O HIV-1 recombinant peptides(recombinant proteins) as can be obtained by conventional methods andhaving the sequences described, for example in application EP 0,591,914.Such compositions are also within the scope of the present invention.

The synthetic peptides of the invention can also be used in combinationwith other HIV-2 and/or HIV-1 recombinant (recombinant proteins) orsynthetic peptides, such as the peptides described in patentapplications or patents EP 0,387,914, EP 0,239,425, EP 0,220,273 or EP0,267,802. This list of patent applications or patents is not exhaustiveand is given by way of example.

The compositions containing one or more synthetic peptides of formula(I) and one or more HIV-1 or HIV-2 recombinant or synthetic peptidesfind application in diagnosis for the screening of patients infectedwith various HIV retroviruses. These compositions also form part of thepresent invention.

Immunoassay methods using one or more synthetic peptides of formula (I),alone or in combination with group O HIV-1 recombinant peptides or HIV-1and/or HIV-2 recombinant or synthetic peptides, also form part of theinvention.

The invention also relates to kits, for carrying out immunoassays, whichinclude a peptide of formula (I) or a composition which contains atleast one peptide of formula (I).

The following examples illustrate the invention and are given with nolimitation being implied.

EXAMPLE 1

Preparation of a Compound According to the Invention: PEPTIDE No. 2 (3B)LLSLWGCRGRLVCYTSVQWNET or Leu Leu Ser Leu Trp Gly Cys Arg Gly Arg LeuVal  1              5                  10 Cys Tyr Thr Ser Val Gln TrpAsn Glu Thr          15                 20    22

This peptide was synthesized on a solid phase. The technique developedin 1963 by Merrifield (J. Am. Chem. Soc. (1963) X, pp. 2149-2154)consists in attaching the first amino acid onto a polymeric solidsupport (resin) by its acid functional group and in extending thepeptide sequence from this first amino acid, the peptide beingsynthesized remaining anchored on the resin.

For the synthesis of peptide No. 2, there were used, as synthesizer, thesynthesizer “9050 Plus Pep Synthesizer” and as resin, the resin Fmoc Thr(OtBu) PEG PS.

The various steps of the synthesis are summarized in Table I below:TABLE I EQ NUMBER - AMINO NH₂ DURATION ACID PROTEC- SIDE COUPLING OFRESIDUE TION PROTECTION METHOD COUPLING Glu Fmoc OtBu DIPCDI/HOBt 5 eq -30 min Asn Fmoc Trt DIPCDI/HOBt 5 eq - 30 min Trp Fmoc Boc DIPCDI/HOBt 5eq - 30 min Gln Fmoc Trt DIPCDI/HOBt 5 eq - 30 min Val Fmoc DIPCDI/HOBt5 eq - 30 min Ser Fmoc tBu DIPCDI/HOBt 5 eq - 30 min Thr Fmoc tBuDIPCDI/HOBt 5 eq - 30 min Tyr Fmoc tBu DIPCDI/HOBt 5 eq - 30 min CysFmoc Trt DIPCDI/HOBt 5 eq - 30 min Val Fmoc DIPCDI/HOBt 5 eq - 30 minLeu Fmoc DIPCDI/HOBt 5 eq - 30 min Arg Fmoc Pbf DIPCDI/HOBt 5 eq - 30min Gly Fmoc DIPCDI/HOBt 5 eq - 30 min Arg Fmoc Pbf DIPCDI/HOBt 5 eq -30 min Cys Fmoc Trt DIPCDI/HOBt 5 eq - 30 min Gly Fmoc DIPCDI/HOBt 5eq - 30 min Trp Fmoc Boc DIPCDI/HOBt 5 eq - 30 min Leu Fmoc DIPCDI/HOBt5 eq - 30 min Ser Fmoc tBu DIPCDI/HOBt 5 eq - 30 min Leu FmocDIPCDI/HOBt 5 eq - 30 min Leu Fmoc DIPCDI/HOBt 5 eq - 30 min

At the end of the synthesis, the resin was washed with dimethylformamideand then dichloromethane and dried under vacuum.

Next, the resin was treated with the K reagent (82% trifluoroaceticacid; 5% phenol; 5% water; 5% thioanisole; 3% ethanedithiol). PeptideNo. 2 (3B), isolated by precipitation with the aid of diethyl ether, wasthen rinsed with the same solvent. 140 mg of peptide No. 2 (3B) werethus obtained.

Peptide No. 2 (3B) was then purified by reversed phase liquidchromatography. The Bondapak C-18 phase was used as solid phase. Thepeptide was eluted by forming a linear gradient between two buffersolutions, the first which is essentially aqueous (for example water-TFA0.1%) and the second which is rather organic (for example a mixturecontaining: 60% acetonitrile, 39.92% water and 0.08% TFA). The purefractions collected were combined, concentrated under vacuum andfreeze-dried.

For the cyclization, the purified synthetic peptide thus obtained wasdissolved in an ammonium acetate solution (10 mM). The pH was adjustedto 8.5 by addition of 1 M ammonium hydroxide. The solution wasvigorously stirred. The cyclization was complete after 18 hours. The pHwas then reduced to 6 by addition of acetic acid. The cyclized peptidewas freeze-dried and then purified by reversed phase liquidchromatography as described above.

Preparation of a Compound According to the Invention: PEPTIDE No. 15(22B)

This peptide was synthesized as peptide No. 2 (3B), but using as resinthe resin FmocPAL PEG-PS.

The various steps of the synthesis are summarized in Table II below:TABLE II EQ NUMBER - AMINO NH₂ DURATION ACID PROTEC- SIDE COUPLING OFRESIDUE TION PROTECTION METHOD COUPLING Val Fmoc DIPCDI/HOBt 5 eq - 45mn Ser Fmoc tBu DIPCDI/HOBt 5 eq - 45 mn Thr Fmoc tBu DIPCDI/HOBt 5 eq -45 mn Tyr Fmoc tBu DIPCDI/HOBt 5 eq - 45 mn Cys Fmoc Trt DIPCDI/HOBt 5eq - 45 mn Val Fmoc DIPCDI/HOBt 5 eq - 45 mn Leu Fmoc DIPCDI/HOBt 5 eq -45 mn Arg Fmoc Pbf DIPCDI/HOBt 5 eq - 45 mn Gly Fmoc DIPCDI/HOBt 5 eq -45 mn Lys Fmoc Boc DIPCDI/HOBt 5 eq - 45 mn Cys Fmoc Trt DIPCDI/HOBt 5eq - 45 mn Gly Fmoc DIPCDI/HOBt 5 eq - 45 mn Trp Fmoc Boc DIPCDI/HOBt 5eq - 45 mn Ser Fmoc tBu DIPCDI/HOBt 5 eq - 45 mn Asn Fmoc TrtDIPCDI/HOBt 5 eq - 45 mn Leu Fmoc DIPCDI/HOBt 5 eq - 45 mn Leu FmocDIPCDI/HOBt 5 eq - 45 mn Arg Fmoc Pbf DIPCDI/HOBt 5 eq - 45 mn Gln FmocTrt DIPCDI/HOBt 5 eq - 45 mn Gln Fmoc Trt DIPCDI/HOBt 5 eq - 45 mn AsnFmoc Trt DIPCDI/HOBt 5 eq - 45 mn Leu Fmoc DIPCDI/HOBt 5 eq - 45 mn LeuFmoc DIPCDI/HOBt 5 eq - 45 mn Thr Fmoc tBu DIPCDI/HOBt 5 eq - 45 mn GluFmoc OtBu DIPCDI/HOBt 5 eq - 45 mn Leu Fmoc DIPCDI/HOBt 5 eq - 45 mn AlaFmoc DIPCDI/HOBt 5 eq - 45 mn Arg Fmoc Pbf DIPCDI/HOBt 5 eq - 45 mn

At the end of the synthesis, the resin was washed withdimethylformamide, followed by dichloromethane and dried under vacuum.

Next, the resin was treated with the K reagent (82% trifluoroaceticacid; 5% phenol; 5% water; 5% thioanisole; 3% ethanedithiol). Thepeptide No. 7 (22B) isolated by precipitation with the aid of diethylether was then rinsed with the same solvent. 140 mg of peptide No. 15(22B) were thus obtained.

Peptide No. 15 (22B) was then purified by reversed phase liquidchromatography and then cyclized, freeze-dried and purified as describedabove for peptide No. 2 (3B).

In the same manner, and using the appropriate resins and amino acids,the other compounds of the invention were synthesized.

Table III indicates the molecular weight of some peptides of formula(I), in non-cyclized form, evaluated by mass spectrometry. TABLE IIIPeptide No. Molecular weight (Daltons) 1 (2B) 2512 2 (3B) 2583 3 (4B)2528 4 (5B) 2586 5 (6B) 2527  9 (12B) 1772 10 (14B) 1799 11 (18B) 375212 (19B) 3778 13 (20B) 3780 14 (21B) 2538 15 (22B) 3222 16 (23B) 1941

EXAMPLE 2

Evaluation of the Immunoreactivity of the Peptides According to theInvention by the Immunoenzymatic Test: Test No. 1

The sera used ESS, DUR, VAU and HAD are sera from French patientsinfected with group O HIV-1 retroviruses. The other serum samples frompatients infected with group O HIV-1 retroviruses were obtained by theYaounde Pasteur Centre in Cameroon and were serotyped group O accordingto the serological algorithm described in AIDS (1977), 11 pp 445-453.

The HIV-negative sera (n=48) were obtained from healthy volunteers.

The synthetic peptides used were dissolved in water at a concentrationof 1 mg/ml (stock solution). For the solid phase sensitization step(coating), 110 μl of a solution at 2 μg/ml of each peptide (obtained bydiluting the stock solution with 0.1 M carbonate buffer solution) wereadded to each well of the microtitre plates Microtiter™ (NUNC). Afterincubating overnight at room temperature, the microplates were firstwashed with a Tris-NaCl buffer solution pH 7.4 containing 0.1% of Tween®20 and 0.001% sodium merthiolate, and then saturated with a PBS solutioncontaining 0.5% of Régilait™ (dried skimmed milk). After aspirating thesaturating solution, the plates were heated for 10 min at 50° C.

The serum samples were diluted ⅕ with a skimmed milk solution (citratebuffer supplemented with 0.01% of phenol red, 0.25% of chloroform and0.25% of Kathon®), deposited in the wells of the plates and incubatedfor 30 min at 40° C.

After washing with a Tris-NaCl buffer solution pH 7.4 containing 0.1% ofTween® 20 and 0.001% of sodium merthiolate, 100 μl of a solution ofconjugate consisting of horseradish peroxidase-labelled anti-human IgGand IgM goat antibodies, containing as preservative 0.01% of sodiummerthiolate, in solution in a citrate buffer solution supplemented with30% glycerol and 25% normal foetal calf serum, were added to each platewell and then the plates were incubated for 30 min at 40° C.

After washing with a Tris-NaCl buffer solution pH 7.4 containing 0.1% ofTween® 20 and 0.001% of sodium merthiolate, the colour was developed byadding, to each well, 100 μl of O-phenylenediamine in solution inhydrogen peroxide. The microplates were then incubated for 30 min atroom temperature and in the dark. The coloured reaction was then stoppedby addition of 100 μl of 4N sulphuric acid. The absorbance (A) wasdetermined at 490 and 620 nm.

The relative absorbance (A490-A620) read in each well is proportional tothe immunoreactivity of each peptide. It indicates the ability of eachpeptide to react with the biological sample with which the test iscarried out. The cut-off value was determined as being an absorbanceequal to 0.15. It corresponds to the mean of the negative values (n=48)plus 12 standard deviations.

The reactivity of the peptides of the invention (peptide No. 3 (4B),peptide No. 2 (3B) and peptide No. 1 (2B), all in cyclized form), wascompared with that of two synthetic peptides having, as sequence, aportion of the natural sequence of the envelope (env) of the VAU isolate(group O HIV-1 retrovirus) and comprising an immunodominant epitope ofgp41.

These two peptides have the following sequence: VAU 22 AA Leu Leu AsnLeu Trp Gly Cys Lys Asn Arg Ala Ile  1               5                  10 Cys Tyr Thr Ser Val Lys Trp AsnLys Thr           15                20    22 VAU 35 AA Arg Leu Leu AlaLeu Glu Thr Phe Ile Glu Glu Asn   1               5                  10Glu Leu Leu Asn Leu Trp Gly Cys Lys Asn Arg Ala          15                20 Ile Cys Tyr Thr Ser Val Lys Trp Asn LysThr 25                  30                  35

For the study, these peptides were used in cyclized form. The results ofthis study are indicated in Table IV. TABLE IV ABSORBANCE PEPTIDEPEPTIDE PEPTIDE VAU 35 SERUM No. 3 (4B) No. 2 (3B) No. 1 (2B) VAU 22 AAAA ESS* >** > 2.494 > > DUR* > > > 0.118 0.872 HAD > 0.518 0.041 0.7890.871 VAU* 1.342 > > > > 3935 > 0.893 0.307 0.138 0.227 6891 > 0.6140.062 0.359 0.496 6512* 0.746 0.785 > 0.120 0.174 1105* 1.421 1.031 >0.099 0.129 4021* 0.430 0.119 > 0.050 1.957 5969* > 0.282 > 2.491 >2700 > 0.274 > > > 5453 0.555 0.081 > 1.267 1.482 5931 > > > 0.202 2.2253136 > 0.992 0.302 > > 3653 1.352 > 0.044 1.441 1.322 2352 > > 0.205 > >3016 > > 0.243 > > 3302 > > 0.386 > > 2294 > > 0.447 > > 3771 > >0.544 > > 1581 > > > 1.112 0.894 5373 > > > 1.359 0.856 7443 > > > 0.9200.574 3637 > > > 0.779 1.647 6295* 1.718 1.063 > 0.972 > 6689*0.710 > > > > 1754 > > > 1.263 1.948 4489* > > > 1.318 1.718 4364 > >1.382 > > 3884* > > 1.839 > > 3529 > > 1.803 > > 3482 2.402 > 1.473 > >1702 > > 1.162 > > 6487 > 1.017 2.687 2.889 2.891 5164 > > > > >5766* > > > > > 3945 > > > > > 4434 > > > 2.273 > 4288* > > 2.802 2.337N.T.*** 6782 > 2.091 2.462 2.190 2.214 2313 > > > > > 2312 > > > > >1062 > > > > >  402 > > > > >  134 > > > > > 7120 > > > > >7212 > > > > > 6976* > > > > > 3600* > > 2.743 > > 3236 > > > > >3235 > > > > > 2551 > > > > > 5270* > > > > > 5210 > > > > >5149* > > > > > 4477 > > > 2.511 > 3891 > > 2.780 > > 3627* > >2.910 > > 7258* > > 2.477 > > 7007 2.136 2.334 > > 2.151 6697 > > > > >6998 > > > > > 6627 > > > > > 6198* > > > > > 6165 > > 2.714 > >7439 > > > > > 7297* > > > > > 6111 > > > > >  625 > > > > 2.885*serotypes/genotypes**> = signal greater than the reading capacity of the spectrophotometer.***Not tested

The results of Table IV demonstrate that peptide No. 3 (4B) exhibits thebest performance with regard to that noted for the other peptides. Thispeptide allows the best discrimination between the sera of patientsinfected with group O HIV-1 retroviruses compared with the two peptideshaving a portion of the sequence of the VAU isolate corresponding to theimmunodominant epitope of gp41. Moreover, peptide No. 2 (3B) and No. 1(2B) of the invention are more immunoreactive than the VAU 22 M peptidewhich comprises the same number of amino acids.

EXAMPLE 3

Evaluation, by an Immunoenzymatic Test, of the Immuno-Reactivity of thePeptides According to the Invention: Test No. 2

The serum samples from patients infected with group O HIV-1 retroviruseswere obtained by the Yaounde Pasteur Centre in Cameroon and wereserotyped group O according to the serological algorithm described inAIDS (1977), 11, pp. 445-453. A genotyped sample (Maryland) is obtainedfrom the United States. These samples were previously diluted innegative human serum at the dilutions given in Table V, in order to havea sufficient volume for the different immunoreactivity tests.

The synthetic peptides used were dissolved in water at a concentrationof 1 mg/ml (stock solution). For the solid phase sensitization step(“coating”), the procedure was carried out as described for Example 2.

The serum samples were diluted ⅕ with a skimmed milk solution (citratebuffer supplemented with 0.01% of phenol red, 0.25% of chloroform and0.25% of Kathon®), deposited in the wells of plates and incubated for 30min at 40° C.

After washing with a Tris-NaCl buffer solution pH 7.4 containing 0.1% ofTween® 20 and 0.001% of sodium merthiolate, 100 μl of a solution ofconjugate consisting of horseradish peroxidase-labelled anti-human IgGand IgM goat antibodies, containing as preservative 0.01% of sodiummerthiolate, in solution in a citrate buffer solution supplemented with30% glycerol and 25% normal foetal calf serum, were added to each wellof the plates and then they were incubated for 30 min at 40° C.

After washing with a Tris-NaCl buffer solution pH 7.4 containing 0.1% ofTween® 20 and 0.001% of sodium merthiolate, the colour was developed asdescribed in Example 2.

The relative absorbance (OD) (A490-A620) read in each well isproportional to the immunoreactivity of each peptide. It indicates theability of each peptide to react with the biological sample with whichthe test is carried out.

The reactivity of the peptides of the invention, peptides No. 10 (14B),No. 11 (18B), No. 12 (19B), No. 14 (21B), No. 15 (22B), No. 16 (23B) allin cyclized form, was compared with that of three homologous syntheticpeptides having, as sequence, a portion of the natural sequence of theenvelope (env) of a group O HIV-1 retrovirus. These peptides are twopeptides derived from the VAU isolate—the peptide VAU 22 AA and thepeptide VAU 35 AA—and the peptide MVP 5180 (designated “MVP 5180” inTable V). The peptides VAU 22 AA and VAU 35 M (whose structure isindicated in Example 2) and the peptide MVP 5180 comprise animmunodominant epitope of gp41.

All these peptides were used in cyclized form. The sequence of the MVP5180 peptide is the following: MVP 5180 Arg Leu Gln Ala Leu Glu Thr LeuIle Gln Asn Gln   1               5                  10 Gln Arg Leu AsnLeu Trp Gly Cys Lys Gly Lys Leu          15                 20 Ile CysTyr Thr Ser Val Lys Trp Asn Thr Ser 25                 30                 35

The results of this study are indicated in Table V. TABLE V PEPTIDES*MVP VAU VAU No. 10 No. 11 No. 12 No. 14 No. 15 No. 16 5180 35 AA 22 AASERUM ABSORBANCE (OD) 4280 0.022 0.686 0.201 0.286 0.689 0.033 0.3820.013 0.021 at 1/50 NGO 0.067 0.335 0.193 0.157 0.315 0.110 0.184 0.0550.040 at 1/50 NJEM 0.032 0.811 0.391 0.277 0.939 0.025 0.146 0.159 0.024at 1/100 MBASSI 1.217 1.150 0.747 2.134 2.010 2.683 0.248 0.120 0.257 at1/100 WANG 0.698 0.234 0.124 2.397 2.680 1.290 0.075 0.025 0.041 at 1/50258 OUDI 0.587 0.373 0.226 0.764 1.184 1.692 0.116 0.058 0.100 at 1/100DO15 1.613 0.859 1.286 3.357 3.693 3.038 0.673 0.036 0.075 at 1/100 DJOU1.268 0.482 0.419 1.998 2.088 2.166 0.203 0.022 0.042 at 1/100 36000.482 0.360 0.249 0.716 0.801 0.933 0.206 0.025 0.058 at 1/100 36131.108 0.837 0.773 1.508 1.627 1.679 0.478 0.250 0.396 at ¼00 6111 0.5960.348 0.202 0.850 1.207 1.009 0.226 0.087 0.180 at 1/100 625 0.838 0.3380.264 2.045 2.122 1.791 0.202 0.069 0.165 at 1/50 Maryland 0.524 0.3700.285 0.734 0.844 1.229 0.241 0.054 0.168 at ¼00 3653 0.347 0.337 0.2470.072 0.380 0.406 0.401 0.021 0.310 at 1/10SOLID PHASE*: PEPTIDE 2 μg/ml

For each peptide tested, the samples were arranged into four classes (a,b, c and d) corresponding to various levels of relative absorbance readat the wavelengths A492-A620:

-   -   for a: OD<0.100,    -   for b: 0.100<OD<0.500,    -   for c: 0.500<OD<1.000,    -   for d: OD>1.000,        thus making it possible to evaluate the degree of        immunoreactivity of the peptides. The most immunoreactive        peptides are those for which the highest number of samples is        found in the classes corresponding to the highest absorbance        values.

The results are indicated in Table VI. TABLE VI PEPTIDES* MVP VAU VAUNo. 10 No. 11 No. 12 No. 14 No. 15 No. 16 5180 35 AA 22 AA CLASS Numberof samples A 3 0 0 1 0 2 1 11 7 B 2 9 11 3 2 2 12 3 7 C 5 4 2 4 4 1 1 00 D 4 1 1 6 8 9 0 0 0SOLID PHASE*: PEPTIDE 2 μg/ml

The results show that all the peptides of the invention tested achieve abetter performance in immunoreactivity than the reference peptides ofthe prior art which are derived from natural isolates (MVP 5180, VAU).The peptides of the invention No. 15 (22B), No. 14 (21B), and No. 16(23B) are found to be the most immunoreactive.

EXAMPLE 4

Evaluation of the Immunoreactivity of the Compositions ContainingPeptides According to the Invention by an Immunoenzymatic Test.

For this test, the procedure was carried out according to the protocoldescribed in Example 2 and the same sera were used. The microplates usedwere sensitized either with peptide No. 1 (2B) cyclized, or with peptideNo. 3 (4B) cyclized, or with a composition containing these two peptides(1:1 w/w). Into each well, there were deposited either 100 μl of asolution containing 2 μg/ml of peptide No. 1 (2B), or 100 μl of asolution containing 2 μg/ml of peptide No. 3 (4B), or 100 μl of asolution containing 1 μg/ml of peptide No. 1 (2B) and 1 μg/ml of peptideNo. 3 (4B).

The results of this test are given in Table VII. TABLE VII ABSORBANCEPEPTIDE PEPTIDE PEPTIDE No. 1 (2B) No. 1 (2B) No. 3 (4B) (1 μg/ml) +PEPTIDE SERUM (2 μg/ml) (2 μg/ml) No. 3 (4B) (1 μg/ml) 3529 1.803  >* >1105 > 1.421 > 3891 2.780 > > 3235 > > > 2700 > > > 5931 > > > 39350.307 > > 7443 > > > 1062 > > > 1754 > > > 3136 0.302 > > 6891 0.062 > >5149 > > > 5270 > > > 2551 > > > 3600 2.743 > > 6976 > > > 4489 > > >6165 2.714 > > 6198 > > > 6627 > > > 6998 > > > 6697 > > > 72582.477 > > 3627 2.910 > > 4477 > > > 3771 0.544 > > 1702 1.016 > > 22940.447 > > 2352 0.205 > > 3016 0.243 > > 3302 0.386 > > 3482 1.473 > >3653 0.044 1.322 1.105 4364 1.382 > > 3637 > > > 4288 2.802 > >5969 > > >  258 > > > 6111 > > >  625 > > > 6853 > 2.769 > 31360.302 > > 6689 > 0.710 > 6295 > 1.718 > 4021 > 0.430 2.381 38841.839 > > 6512 > 0.746 > 6487 2.687 > > ESS 2.494 > > HAD 0.041 > >DUR > > >*> = signal greater than the reading capacity of the spectrophotometer.

The results of Table VII demonstrate that the compositions of thepeptides of the invention, when used in diagnosis, allow the detectionof all the sera from patients infected with group O HIV-1 retroviruses.

1-14. (canceled)
 15. Synthetic peptide in linear form or in a formcyclized by means of intercysteine disulphide bridges, having thegeneral formula (I):Δ-Z-TrpGlyCys-Θ-CysTyrThrSer-Ω wherein Δ is selected from the groupconsisting of a biotinyl radical, a biocytinyl radical, a hydrogen atom,an acetyl (CH₃CO—) radical, an aliphatic chain which may contain one ortwo thiol, an aldehyde functional group and an amine functional group, Zrepresents a peptide sequence, selected from the group consisting of thesequences of the formulae:

-Ser-

₂  (II)

₁-Ser  (IV)

-Gln-

₂  (V)

₁-Asn-

2  (VIII) wherein

₁ represents a peptide sequence of 0 to 9 amino acids, and

₂ represents a peptide sequence of 0 to 5 amino acids, Θ represents apeptide sequence of formula (XI):(AA₁)-Gly-(AA₃)-Leu-(AA₅)  (XI): wherein (AA₁) is selected from thegroup consisting of a lysine residue and an arginine residue, (AA₃) isselected from the group consisting of a lysine residue and an arginineresidue, (AA₅) is selected from the group consisting of an isoleucineresidue and a valine residue, provided, however, that Θ never representsthe peptide sequence Lys Gly Lys Leu Ile or Lys Gly Lys Leu Val, Ωattached to the —CO— group of serine, is selected from the groupconsisting of: a hydroxyl (—OH) radical and a peptide sequence offormula (XII):Val-Σ-Ψ  (XII) wherein Σ represents a sequence:(AA₆)-Trp Asn-(AA₇)-(AA₈)  (XIII) wherein: (AA₆) represents an aminoacid different from lysine, (AA₇) represents an amino acid, (AA₈) isselected from the group consisting of a serine and a threonine residue,and Ψ, attached to the —CO— residue of the free AA₈ amino acid, isselected from the group consisting of an OH group and a NH₂ group. 16.Synthetic peptide of formula (I) according to claim 15 wherein Δrepresents an aliphatic chain, the aliphatic chain being selected fromthe group consisting of an alkyl chain of 1 to 6 carbon atoms, analkenyl chain of 2 to 6 carbon atoms, and an aminoalkylcarbonyl chain of2 to 6 carbon atoms.
 17. Synthetic peptide of formula (I) according toclaim 15 wherein Θ is selected from the group consisting of: Arg Gly ArgLeu Val, Arg Gly Lys Leu Ile, Arg Gly Lys Leu Val, and Lys Gly Arg LeuVal.
 18. Synthetic peptide of formula (1) according to claim 15 whereinΩ corresponds to the peptide sequence of formula (XII) and (AA₆) isselected from the group consisting of a glutamine residue and anarginine residue.
 19. Synthetic peptide of formula (I) according toclaim 15 wherein Ω corresponds to the peptide sequence of formula (XII)and (AA₇) is a serine residue.
 20. Synthetic peptide of formula (I)according to claim 15 wherein Ω corresponds to the peptide sequence offormula (XII) and (AA₇) is a threonine residue.
 21. Synthetic peptide offormula (I) according to claim 15 wherein Ω corresponds to the peptidesequence of formula (XII), (AA₇) is a threonine residue, and (AA₈) is aserine residue.
 22. Synthetic peptide of formula (I) according to claim15 wherein Ω corresponds to the peptide sequence of formula (XII), (AA₆)is a glutamine residue or an arginine residue, and (AA₈) is a serineresidue or a threonine residue.
 23. Synthetic peptide of formula (I)according to claim 15 wherein Ω corresponds to the peptide sequence offormula (XII), (AA₆) is a glutamine residue, and (AA₈) is a serineresidue or a threonine residue.
 24. Synthetic peptide of formula (I)according to claim 15 wherein Ω corresponds to the peptide sequence offormula (XII), (AA₆) is an arginine residue, and (AA₈) is a serineresidue or a threonine residue.
 25. Synthetic peptide of formula (I)according to claim 15 wherein Ω corresponds to the peptide sequence offormula (XII) and (AA₈) is a serine residue or a threonine residue. 26.Synthetic peptide of formula (I) according to claim 15 wherein Ωcorresponds to the peptide sequence of formula (XII) and (AA₈) is aserine residue.
 27. Synthetic peptide of formula (I) according to claim15 wherein Ω corresponds to the peptide sequence of formula (XII) and(AA₈) is a threonine residue.
 28. Synthetic peptide of formula (I)according to claim 15 wherein Ω is peptide sequence -Val Gln Trp Asn SerThr T.
 29. Synthetic peptide of formula (1) according to claim 15wherein Z is selected from the group consisting of peptide sequences offormulae: -Leu Leu Ser Leu-, -Leu Leu Ser Ser-, -Leu Leu Asn Ser-, -ArgLeu Asn Ser-, -Ala Leu Glu Thr Leu Leu Gln Asn Gln Gln Leu Leu Asn Ser-,-Ala Leu Glu Thr Leu Leu Gln Asn Gln Gln Leu Leu Asn Ile-, -Leu Asn GlnGln Arg Leu Leu Asn Ser-, and -Arg Ala Leu Glu Thr Leu Leu Asn Gln GlnArg Leu Leu Asn Ser-.